We have identified, in Saccharomyces cerevisiae, a new mechanism of transcriptional regulation that modulates transcription by increasing or decreasing expression levels in conditions of activation or repression, respectively. TRM regulation exerts an effect on genes specifically regulated in response to a diverse range of stimuli including external signals, cell-type, or cell-differentiation processes. TRM increases efficiency of regulation by decreasing the repression over activation ratio. We have selected cis-acting and trans-acting mutations that abolish TRM- mediated transcriptional control and identified two genes, TRM1 and TRM2, required for TRM regulation. We have cloned TRM1 and TRM2 and with these molecular genetic tools, we propose to i) identify proteins that function in the TRM system, ii) determine the molecular basis of transcriptional modulation, and iii) identify TRM1 and TRM2 domains that mediate the protein-protein interactions required for repression and/or activation of transcription. Negative phenotypes available in wild-type (Trm+) or mutant (Trm-) cells will allow positive selection for mutations that increase or decrease TRM function. We will genetically identify proteins that interact with TRM1 and TRM2 by positive selection for extragenic suppressors of trm1 and/or trm2 mutations. Genetic and molecular analysis of the genes encoding these suppressors will provide the necessary tools to analyze the TRM system. We will genetically identify proteins that confer TRM repression through their interaction with upstream repressor sequences (URS) in the promoters of TRM-repressible genes. Once genetically identified, the genes encoding these repressors will be analyzed. We have found that the expression of E1A confers a toxic, sublethal phenotype to TRm- but not Trm+ cells. This is the first time, to our knowledge, that a phenotype has been observed in connection with E1A expression in S. cerevisiae. It provides a unique opportunity to pursue molecular genetic strategies designed to analyze E1A function in this eukaryote. In addition, by screening mammalian cDNA libraries for clones that suppress E1A-mediated toxicity in yeast, we propose to identify proteins, like Rb, that interact with E1A.